Summary: Pneumococcal polysaccharide antigens induce type-specific antibodies that enhance opsonization, phagocytosis, and killing of pneumococci by leukocytes and other phagocytic cells. The licensed pneumococcal conjugate vaccine, Prevnar, contains polysaccharides from the seven most prevalent serotypes that cause invasive pneumococcal disease in children. The serotype 19F conjugate included in the vaccine induced higher antibody levels in clinical studies than several of the other serotypes, yet 19F was isolated from a high percentage of otitis media vaccine failures, leading to our investigation of possible alterations in the epitopes expressed by 19F conjugates prepared using reductive amination. We have now screened 113 human sera from different NIH blood bank donors by ELISA for antibodies to 19F using the native (unmodified) PS, 19F conjugated to tyramine using a cyanylation method, and 19F conjugated to human serum albumin (HSA) prepared by reductive amination, the method used for the licensed vaccine. The binding ratio of Pn 19F-HSA to Pn 19F-native was determined. A ratio greater than 1 indicates more binding to the conjugate epitope. Results show that there are 44/113 (39.82 %) with a ratio of <0.5, 55/113 (48.67%) with a ratio of 0.5-2.0, and 14/113 (12.39%) with a ratio of >2.0. A ratio of 0.5-2.0 indicates no significant difference in binding between the 2 antigens, >2.0 ratio means there is two-fold more binding in Pn 19F-HSA than in Pn 19F- native PS, and a ratio of <0.5 means there is twofold more binding in Pn 19F-native than the Pn 19F-HSA antigen in the ELISA. Functional activity of these antibodies to the different19F epitopes was determined by the opsonophagocytosis assay using human leukocytes as the effector cells. There are serum samples (5) that have high antibody levels to the 19F-HSA (6.0-15.5 ug/ml) and low levels of antibodies to the native 19F PS (0.34-2.4ug/ml) that were found to be not opsonic to Pn 19F strain. On the other hand, there are serum samples (4) that have as much as 5.0-7.0 ug/ml to the native PS and yet, not opsonic to the 19F strain also. These antibodies need to be examined further, for specificity and avidity for example, to understand why these antibodies are not functional. Additional 34 post-vaccination sera from a vaccine made by another conjugation method (not by reductive amination) were also examined. Only 2/34 (5.9%) of the samples have a ratio of >2.0 (2.9 and 3.01) compared to 7/34(20.6%) of the samples that have a ratio of <0.5 (0.26-0.49). 73.5% of the samples (25/34) have a ratio of 0.5-2.0. Likewise, the functional activity of these sera needs to be examined by opsonophagocytosis assay. Sub-classes of IgG (IgG1, IgG2, and IgG3) in IVIG (Immunoglobulin) preparations from 3 different manufacturers (in collaboration with B. Golding laboratory) were assayed for antibody levels in 5 Pn serotype (4,6B, 9V, 14 and 19F) by ELISA and functional activity of these antibodies were examined by opsonophagocytosis assay. There was a predominance of IgG2 to the different serotypes tested and these antibodies were functional by opsonophagocytosis assay.